Objectives: To date, the scientific source materials usually focus on microbial contamination of the museum or library collections themselves, while the exposure of persons who professionally deal with this type of objects in cultural heritage conservation laboratories is ignored. Material and Methods: The study was carried out in 9 naturally ventilated conservation laboratories with no history of water damage. Viable (understood as culturable) bioaerosol stationary samples were collected in both outdoor and indoor environments using 6-stage Andersen impactor. Simultaneously, stationary and personal indoor bioaerosol measurements were carried out using both Gesamtstaubprobenahme an der Person (GSP) and Button filter samplers. These measurements were complemented by evaluation of microbial content in the dust settled on conserved works of art. All impactor, filter, and settled dust samples were quantitatively examined to obtain viable and total concentrations of bacteria and fungi. All isolated microbial strains were taxonomically identified. Results: At workplaces, the concentrations of viable microorganisms in air were below 2000 cfu/m³ and accounted for not more than 5.5% of total microbiota. The study showed that quantitative assessment of viable bioaerosol can be made with an Andersen impactor as well as by using Button and GSP filter samplers, irrespective of whether they are applied for personal or stationary measurements. Compared to the impactor, however, the use of filter samplers for microbial contamination monitoring substantially limits the scope of qualitative information which can be obtained. Size distribution analysis revealed that the largest “load” of microorganisms can penetrate into the respiratory tract between the trachea and terminal bronchi, and thereby may be responsible for allergic inflammations in exposed workers. Conclusions: The precise assessment of microbial hazards in conservation laboratories should comprise control of both viable and total particle counts. The hermetization of such workplaces and control of relative humidity should be implemented and maintained to assure proper hygienic conditions.